1. Field of the Invention
The invention relates to the fields of medicine and cell biology. More specifically, the invention relates to the fields of drug discovery and dermatology, particularly the biology of skin pigementation.
2. Description of Related Art
Melanin is a dark pigment found in plants and animals that protects against ultraviolet radiation and provides decoration in the skin, eyes, hair, and fur of animals (reviewed in Riley, P. A., 1997, Int. J. Biochem. Cell Biol. 11:1235-39). There are two different types of melanin: brown/black eumelanin and yellow/red pheomelanin. Melanocytes are cells of the epidermis specialized to produce melanin. A sophisticated intercellular signaling system determines whether an individual melanocyte will produce eumelanin or pheomelanin (reviewed in Brilliant, M. H. and Barsh, G. S., 1998, in The Pigmentary System: Physiology and Pathophysiology, 217-29, Oxford University, New York (Nordlund, J. J. et al., eds)).
Melanocytes synthesize melanin inside of specialized organelles called melanosomes (reviewed in Orlow, S. J., 1998, in The Pigmentary System: Physiology and Pathophysiology, 97-106, Oxford University, New York (Nordlund, J. J. et al., eds)). Melanosomes are formed by the fusion of two types of vesicles. One type of vesicle, called a premelanosome, apparently derives directly from either the smooth endoplasmic reticulum or the trans-Golgi network. The other type of vesicle derives from the trans-Golgi network. Each of these types of vesicles contributes proteins to the melanosome necessary for its function.
Defects in the production of melanin result in pigmentation deficiencies such as albinism. Genetic analysis of abnormally pigmented strains of mice has identified more than 60 genes necessary for the normal production of melanin (reviewed in Silvers, W. K., 1979, The Coat Colors of Mice: A Model for Mammalian Gene Action and Interaction, Springer-Verlag, Basel). One of these genes encodes the enzyme tyrosinase. Tyrosinase protein is a multi-functional enzyme that catalyzes several steps in the production of melanin; tyrosinase activities include the rate-limiting steps of converting tyrosine to dihydroxyphenylalanine (DOPA), and DOPA to dopaquinone (reviewed in Lerner, A. B., and Fitzpatrick, T. B., 1950, Physiol. Rev. 30:91-126), as well as the oxidation of 5,6-dihydroxyindole to 5,6-indolequinone (Korner and Pawelek, 1982, Science 217:1163-1165). Both humans and mice lacking tyrosinase activity suffer a severe form of albinism.
Two tyrosinase-related proteins (TRP-1, encoded by the mouse brown gene, and TRP-2, encoded by the mouse slaty gene) also are important for melanogenesis (reviewed in Hearing, V. J., 1993, Am. J. Hum. Genet. 52:1-7). Each of the TRP proteins shares about 40% sequence identity with tyrosinase and with each other. Each of these three enzymes (tyrosinase, TRP-1 and TRP-2) is predicted to contain one transmembrane domain. Together, they form a high molecular weight complex associated with the melanosomal membrane (Orlow, S. J., et al., 1994, J. Invest. Dermatol. 103:196-201).
Another protein that is important for the production of melanin is the P protein. In mice, it is the product of the pink-eye dilution (p) gene. In humans, it is the product of the P gene. p-null mice produce significantly less melanin than wild-type mice (Silvers, supra). A wild-type human P gene, but not a mutant human P gene, can complement the hypopigmented phenotype of p-null mouse melanocytes (Sviderskaya, E. V., et al., 1997, J. Invest. Dermatol. 108:30-34). P protein is apparently needed for the production of eumelanin, but not of pheomelanin (Lamoreux, M. L., et al., 1995, Pigment. Cell. Res. 8:263-70).
Tyrosinase positive oculocutaneous albinism (Ty-pos OCA) or type 2 oculocutaneous albinism (OCA2) is the most common form of albinism worldwide. It results from mutations at the pink-eyed dilution gene (P) (King, R A. et al. (1995) Scriver, C R. et al. (eds) The Metabolic Basis of Inherited Disease, McGraw-Hill, New York. pp 4353-4392; Ramsay, M. et al. (1992) Am. J. Hum. Genet. 51:879-84; Rinchik, E M. et al. (1993) Nature 361: 72-76). Affected individuals have hypopigmented skin, hair and eyes (Manga P. et al. (1999) J. Dermatol. 26:738-47), and are thus at increased risk of developing UV-induced carcinomas (Kromberg, J G. et al. (1989) Clin. Genet. 36:43-52).
The p gene product (p), is predicted to have 12 transmembrane domains (Gardner, J M. et al. (1992) Science 257:1121-1124; Rinchik, E M. et al. (1993) Nature 361:72-76), and is present in melanocytes, the site of melanin synthesis (Rosemblat, S. et al. (1994) Proc. Natl. Acad. Sci. (USA) 91:12071-12075). While p appears to be involved exclusively in eumelanin synthesis (Russell, E S. (1949) Genetics 34:146-166), a precise function is yet to be assigned to the gene.
Several authors have suggested that P protein acts as a tyrosine transporter by pumping tyrosine into the melanosome where it is converted into melanin by tyrosinase activity (see, e.g., Rinchik, E. M., et al, 1993, Nature 361:72-76). First, the P protein bears some resemblance to transport proteins found in prokaryotes. Second, cultured p-null mutant mouse melanocytes, which produce much less melanin than cultured wild-type mouse melanocytes, make increased levels of melanin when high concentrations of tyrosine are added to the cells growth medium (Sviderskaya, E. V., et al., supra; Rosemblat, S. et al., 1998, Exp. Cell Res. 239:344-52). However, contradicting this suggestion, it has been found that tyrosine uptake by melanosomes is virtually the same in p-null and wild-type melanocytes (Gahl, W. A. et al., 1995, Pigment. Cell. Res. 8:229-233). This observation has led other authors to hypothesize that P protein is necessary for the transport into melanosomes of some other small molecule necessary for melanogenesis (summarized in Brilliant, M. H. and Barsh, G. S., 1998, supra).
Other authors have speculated that P protein plays a structural role in melanosomes (Lamoreux, M. L, et al., supra). The integrity of melanosomes is compromised in cells lacking P protein. Tyrosinase activity, and therefore melanin production, is greatly decreased in these defective melanosomes. Specifically, tyrosinase activity levels in melanocyte extracts of skin and eyes from p-null mice are lower than such extracts from wild-type mice (Lamoreux, M. L., et al., supra Chiu, E., et al, 1993, Exp. Eye Res. 57:301-05). Moreover, levels of tyrosinase, TRP-1 and TRP-2 proteins are lower in p-null tissue extracts than in wild-type extracts (Rosemblat, S. et al., 1998, supra). Additionally, a much greater percentage of tyrosinase, TRP-1, and TRP-2 proteins are found in their monomeric forms, rather than as part of a high molecular weight complex, in p-null tissue extracts than in wild-type extracts (Lamoreux, M. L., et al., supra; Chiu, E., et al., supra), and tyrosinase, TRP-1, and TRP-2 are all rapidly degraded in the ocular tissue of p-null mice (Chiu, E., et al., supra). Finally, several authors have observed that melanosomes in p-null tissues and cultured melanocytes are abnormal (Russell, E. S., 1949, Genetics 34:146-66; Rosemblat, S. et al., 1998, supra). In p-mutant melanocytes from mouse eye, very few melanosomes are observed (Orlow, S. J. and Brilliant, M. H., 1999, Exp. Eye Res. 68:147-54). In cultured mutant melanocytes, a greater than normal number of melanosomes is present, but they are smaller than those seen in wild-type melanocytes (Rosemblat, S. et al., 1998, supra).
Thus, although P protein is known to be critical for the production of normal amounts of melanin in the skin, hair and eyes, the function of the P protein in this process has remained elusive. Instead, researchers have looked to other molecular targets for inhibition studies. For example, tyrosinase's well-characterized enzymatic activity, amenability to biochemical analysis, and pivotal role in melanogenesis have made it an inviting target for inhibition studies (see, e.g., Tasaka, K, et al., 1998, Meth. Find. Exp. Clin. Pharmacol. 20:99-109; lida, K, et al., 1995, Planta Med. 61:425-28; Reish, O., et al., 1995, Am. J. Hum. Genet 57:127-32; Shirota, S., et al, 1994, Biol. Pharm. Bull. 17:266-69; Kameyama, K., et al., 1989, Differentiation 42:28-36). Researchers have also focused on the effects of intercellular signaling molecules on melanogenesis (see, e.g., Furumura, M. et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:737478; Sakai, C., et al., 1997, EMBO J. 16:3544-52; McLeod, S. D. et al., 1995, J. Endocrinol. 146:439-47).
Pigmentation disorders of mammals and Drosophila are known and in some cases have been linked to specific defects in proteins involved in intracellular protein trafficking (Spritz (1999) Trends Genet 15:337-340). For example, the hypopigmentation associated with Hermansky-Pudlak syndrome (HPS) appears to be related to the regulation of protein trafficking by the AP3 protein. (Dell'Angelica et al. (1999) Mol. Cell 3:11-21).
For many individuals of all ages, the inappropriate production or overproduction of melanin is a cosmetic problem. By way of example, many children develop freckles after exposure to the sun, and for individuals in middle or advanced age, chloasma, freckles, and pigmentary deposits after sunburn tend to occur or increase in frequency. In addition, these pigment deposits do not disappear quickly and are more likely to become permanent with advancing age.
A number of products have been developed to effect a decrease in skin pigmentation. One such product contains hydroquinone, a well known active substance for skin de-pigmentation (e.g., see U.S. Pat. No. 6,139,854). However, hydroquinone can have serious side effects if applied over a long period of time. For example, the application of hydroquinone to skin may lead to permanent de-pigmentation, and thus to increased photosensitivity of the skin when exposed to ultraviolet light. For that reason, in some countries hydroquinone is only allowed to be used for skin de-pigmentation in limited concentrations, and, in other countries, the product is banned completely for this application.
A variety of other substances have been proposed for the control or inhibition of skin pigmentation. Almost all of these substances work by either bleaching existing pigment or preventing new pigment synthesis by inhibiting the activity of tyrosinase, the principle rate-limiting enzyme in the production of melanin. For example, U.S. Pat. No. 6,123,959 describes the use of aqueous compositions comprising liposomes and at least one competitive inhibitor of an enzyme for the synthesis of melanin in combination with at least one non-competitive inhibitor of an enzyme for the synthesis of melanin. U.S. Pat. No. 6,132,740 describes the use of certain resorcinol derivatives as skin lightening agents. WO 99/64025A1 describes compositions for skin lightening which contain tyrosinase inhibiting extracts from dicotyledonous plant species indigenous to Canada. U.S. Pat. No. 5,580,549 describes an external preparation for skin lightening comprising 2-hydroxybenzoic acid derivatives and salts thereof as inhibitors of tyrosinase. WO 99/09011A1 describes an agent for inhibiting skin erythema and/or skin pigmentation, containing at least one carbostyril derivative and salts thereof. U.S. Pat. Nos. 5,214,028 and 5,389,611 describes lactoferrin hydrolyzates for use as a tyrosinase inhibitory agents.
Despite the development of these and other compositions to lighten skin, there remains a need in the art for the development of less toxic, safer alternatives to skin bleaching and more effective and efficient methods of inhibiting melanin production. The need for new and improved methods for lightening skin is evident in view of the cosmetic industry's estimate that the market for skin lighteners worldwide exceeds well over one billion dollars a year. Thus, there is a continuing need for the development of improved agents that limit or inhibit pigmentation in the skin.